Process

ABSTRACT

A process for the preparation of coated polymer particles containing superparamagnetic crystals, said process comprising reacting porous, surface-functionalized, superparamagnetic crystal-containing polymer particles of diameter 0.5 to 1.8 μm with at least one polyisocyanate and at least one diol or at least one epoxide.

This invention relates to a process for the preparation of coatedmagnetic polymer particles.

Magnetic polymer particles are of general utility in various medical andbiochemical fields, for example as transport vehicles for the deliveryof pharmaceutical products, for diagnostic purposes, for separation andfor synthetic purposes. Such particles rely upon their magneticproperties in order to perform these functions. In diagnostic assayapplications, for example, application of a magnetic field to a samplecontaining an analyte bound to magnetic polymer particles allows theisolation of the analyte without the use of centrifugation or filtrationand in therapeutic applications, for example, application of a magneticfield to the patient may serve to target drug-carrying magnetic polymerparticles to a desired body site.

By magnetic is meant herein that the polymer particles containsuperparamagnetic crystals. Thus the magnetic polymer particles aremagnetically displaceable but are not permanently magnetizable. Manyprocesses for preparing magnetic polymer particles are known, a largenumber of which involve preparing maghemite- or magnetite-containingpolymer particles from pre-formed magnetic iron oxides, e.g. magnetite.Some of processes involved are described in U.S. Pat. No. 4,654,267(Ugelstad) the contents of which are incorporated herein by reference.

Thus U.S. Pat. No. 4,654,267 outlines a number of limitations withregard to the processes which preceded it; these include difficulty inobtaining magnetic particles of similar size and/or of homogeneous oruniform magnetic properties, as well as a more general problem relatingto the difficulty of incorporating magnetic material inside the cavitiesof porous polymer particles.

With deposition taking place principally on the surface, or in largeopen cavities, leaching of magnetic particles, which shortens the usefullifetime of magnetic polymer particles in the applications to which theyare put, was consequently problematic.

In order to overcome these disadvantages, U.S. Pat. No. 4,654,267proposed a preparative method whereby, in its simplest form, porouspolymer particles are impregnated with solutions of iron compoundswhereafter the iron is precipitated, for instance by raising the pHvalue. The precipitated iron compounds may then be converted tosuperparamagnetic iron oxide crystals by heating.

To produce porous magnetic polymer particles having magnetic materialdisposed within the polymer pores, U.S. Pat. No. 4,654,267 advocated theuse of porous polymer particles having surface functional groups whichserve to draw the iron ions into the polymer particles. These functionalgroups could either result from the use of functionalized comonomers inthe production of the polymer or from post-polymerization treatment ofthe polymer to introduce the functional groups, e.g. by coupling to ortransformation of existing groups on the polymer surface.

Whilst the invention disclosed in U.S. Pat. No. 4,654,267 does in partsolve the problem of producing magnetic polymer particles which havemore homogeneous magnetic properties, the problem of leaching of thesuperparamagnetic crystals from the polymer particles remains.

We have now surprisingly discovered that for particles of a certainsize, this problem may be solved and magnetic particles withparticularly suitable surface characteristics may be produced byreacting surface functionalized, magnetic polymer particles with acombination of polyisocyanate/diol or epoxide monomers to produce a“coated” magnetic polymer particle.

Viewed from a first aspect, therefore, the present invention provides aprocess for the preparation of coated polymer particles containingsuperparamagnetic crystals, said process comprising reacting porous,surface-functionalized, superparamagnetic crystal containing polymerparticles of diameter 0.5 to 1.8 μm, more preferably of diameter 0.75 to1.2 μm, especially approximately 1 μm, with at least one, preferably atleast two epoxide compounds.

Viewed from a second aspect, the present invention provides a processfor the preparation of coated polymer particles containingsuperparamagnetic crystals, said process comprising reacting porous,surface-functionalized, superparamagnetic crystal containing polymerparticles of diameter 0.5 to 1.8 μm, e.g. 0.75 to 1.2 μm, especiallyapproximately 1 μm, with at least one polyisocyanate, e.g. diisocyanate,and at least one, preferably at least two, diols.

Preferred diols are polyethylene glycols or are of formulaHO((CH₂)_(m)O)_(n)H (where n is an integer of 1 to 15, e.g. 2 to 10,preferably 2 to 4, and m is an integer of 2 to 6, preferably 2 to 3,most preferably 2). Where only one diol is employed, this is preferablya polyethylene glycol, e.g. polyethylene glycol 300, 400, 500 or 600.

The porous polymer particles used in the process of the invention may beany porous polymer having a functionalized surface, e.g. as described inU.S. Pat. No. 4,654,267.

The surface functionality on the polymer is preferably a group capable,optionally with activation, of reacting with a polyisocyanate or epoxideto covalently bond the polyisocyanate or epoxide to the surface. Mostpreferably the surface is amine functionalized.

The polymer is preferably made from combinations of vinylic polymers,e.g. styrenes, acrylates and/or methacrylates. The polymeric materialmay optionally be crosslinked, for example by incorporation ofcross-linking agents, for example as comonomers, e.g. divinylbenzene(DVB) or ethyleneglycol dimethacrylate. Particles comprising DVB arepreferred.

Appropriate quantities of the cross-linking agents (e.g. comonomers)required will be well known to the skilled man. Preferably the polymeris a cross-linked styrenic polymer (e.g. a styrene-divinylbenzenepolymer, which may be surface functionalized by the use of a nitro-groupcontaining comonomer, e.g. nitro-styrene, and subsequent reduction) or across-linked (meth)acrylic polymer surface functionalized by the use ofan epoxy-group containing comonomer (e.g. glycidylmethacrylate) andsubsequent amination (e.g. by reaction with ethylene diamine).

The superparamagnetic crystals in the polymer particles used in theprocess of the invention may be of any material capable of beingdeposited in superparamagnetic crystalline form in the porous polymerparticles. Magnetic iron oxides, e.g. magnetite or maghemite arepreferred; however the crystals may be of mixed metal oxides or othermagnetic material if desired. The total quantity of crystalline magneticmaterial present is generally more than 1%, preferably more than 3%,desirably more than or equal to 5% (by weight, e.g. up to 40% wt. Thepercentage is calculated on a Fe (or equivalent metal in the case ofmagnetic materials other than iron oxides)-weight basis based upon theoverall dry weight of the coated particles.

Polymer particles according to the present invention will have sizes(i.e. diameters) that are generally in the range 0.5 to 1.8 μm, e.g 0.75to 1.2 μm, preferably 0.9 to 1.1 μm.

Typically the porous particles used will have a surface area of at least15 m5/g (measured by the BET nitrogen absorption method), and morepreferably at least 30 m5/g, e.g. up to 700 m5/g, when corrected to amean particle diameter of 2.7 μm (i.e. multiply surface area by 2.7/MD,where MD is the mean diameter in micrometers). Similarly scaled, theparticle pore volume is preferably at least 0.1 mL/g.

Typically, the polymer particles are spherical and substantiallymonodisperse before they are coated and especially preferably remainspherical and substantially monodisperse once they have been coated.

By substantially monodisperse it is meant that for a plurality ofparticles (e.g. at least 100, more preferably at least 1000) theparticles have a coefficient of variation (CV) of less than 20%, forexample less than 15%, preferably less than 12%, more preferably lessthan 11%, still more preferably less than 10% and most preferably nomore than about 8%, e.g. 2 to 5%. CV is determined in percentage as${CV} = \frac{100 \times \text{standard~~deviation}}{\text{mean}}$where mean is the mean particle diameter and standard deviation is thestandard deviation in particle size. CV is preferably calculated on themain mode, i.e. by fitting a monomodal distribution curve to thedetected particle size distribution. Thus some particles below or abovemode size may be discounted in the calculation which may for example bebased on about 90% of total particle number (of detectable particlesthat is). Such a determination of CV is performable on a Coulter LS 130particle size analyzer.

Of particular utility in the present invention are polymeric particlesdisclosed in WO 99/19375 (Dyno Industrier ASA), WO 00/61648 (DynoSpecialty Polymers AS) made in part from amino styrene as disclosed inWO 01/70825, the contents of which are incorporated herein by reference.

Alternatively, in contrast to the disclosure of WO01/70825,functionalisation of the polymeric material may take place afterpolymerisation by, for example, nitration and subsequent reduction ofthe thus-formed nitro groups to pendant amine groups; or directamination, for example by treatment with amino ethanol. As furtheralternatives, polymeric particles prepared by the well-known Ugelstadtwo-step swelling process and the improvements thereto disclosed in WO00/61647 (Dyno) may be used. Also of use here are polymeric particlesprepared by the processes disclosed in WO99/19375 and WO00/61648. Porouspolymer particles produced according to the processes described in thesepublications may have magnetic particles deposited in their pores bystandard techniques, e.g. as described above. As a further possibility,porous polymer particles may be prepared from nitro styrene and DVB, andmagnetic material introduced as taught in U.S. Pat. No. 4,654,267. Ofall these processes, the use of amino styrene, particularly4-aminostyrene, as monomer or comonomer in the preparation ofamino-bearing polymeric material is preferred. Use of this monomer orcomonomer obviates the need for post-polymerisation nitration andreduction reactions. Moreover, the more predictable nature (homogeneity)of the coating afforded by this process permits a more reliable coatingto be applied.

The reaction of the porous magnetic polymer particle with the epoxidesor polyisocyanates generates a polymer within the pores of the polymerparticles which serves essentially to block these pores physicallyencapsulating the superparamagnetic crystals within the polymerparticles. The resulting “coated” particles then have reduced porosityrelative to the porous starting material. Surprisingly we have foundthat the superparamagnetic crystals appear to catalyse thepolymerization so that the coating forms preferentially in theirvicinity.

If desired, an epoxide polymer coating may be cross-linked, e.g. by useof an isocyanate or diisocyanate in known fashion. Equally if desiredfurther materials may be impregnated into the porous particles eitherbefore the coating polymerization reaction or after coatingpolymerization but before coating polymer cross-linking. Typically suchfurther materials will be radiation emitters or absorbers, e.g.chromophores, fluorophores or radioactively labelled materials.

The particles may be reacted with a single epoxide to form the coating.Suitable epoxides here include glycidol, allylglycidyl ether orglycidylmethacrylate. In particular, a coating reaction involvingglycidylmethacrylate in combination with iron (III) chloride hasprovided advantageous coatings.

However, in a preferred embodiment, the porous polymer particles arereacted with a mixture of epoxides, e.g. 2-6 epoxides, especially 2, 3or 4 epoxides. Of these, diepoxides or polyepoxides preferablyconstitute at least 30 mole %, more preferably at least 45 mole %.

The epoxide compounds used according to the invention preferablycomprise at least one diepoxide, e.g. a combination of two diepoxides orof a monoepoxide and a diepoxide. Preferably, the epoxides contain atleast one ether link and optionally a hydrophobic component, e.g. aC₄₋₁₀ alkylene chain or a phenyl or bisphenyl group. Generally, theepoxides will have a carbon atom content of from 3 to 50, preferably 3to 25.

Typical epoxides that may be used include epichlorohydrin,epibromohydrin, isopropylglycidyl ether, butyl glycidyl ether,allylglycidyl ether, 1,4-butanediol diglycidyl ether(1,4-bis(2,3-epoxypropoxy) butane), neopentylglycol diglycidyl ether,ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether(e.g. of Mw 150 to 1000 g/mol), glycerol diglycidyl ether,glycerolpropoxylate triglycidylether, glycidol, glycidyl methacrylate,and epoxides based on bisphenol A or bisphenol F, e.g.2,2-bis(4-(2,3-epoxypropoxy)phenyl)-propane.

Especially preferred epoxides include2,2-bis(4-(2,3-epoxypropoxy)phenyl)-propane, allylglycidyl ether,1,4-butanediol diglycidyl ether and glycidol. In particular, theepoxides sold under the Araldite trade name are favoured, e.g. AralditeLY-564 (a mixture of 2,2-bis(4-(2,3-epoxypropoxy)phenyl)-propane and1,4-butanediol diglycidyl ether) and Araldite LY-026 (80% pure1,4-butanediol diglycidyl ether).

It is within the scope of the invention for a first coating reaction tobe carried out using a single epoxide and a secondary coating reactionto be carried out using a mixture of at least two epoxides.

In another preferred embodiment, the coating polymer is formed from oneor more (e.g. 1, 2 or 3) polyisocyanates and one or more (e.g. 2, 3 or4) diols. Preferably, one polyisocyante should be employed, e.g. onediisocyanate. Alternatively, a mixture of closely relatedpolyisocyanates can be employed (e.g. Desmodur).

Typical polyisocyanates which may be used include methylenediisocyanate, hexamethylene diisocyanate, 2,4-toluene diisocyanate(2,4-TDI) (and isomers or mixtures thereof), isophorone diisocyanate(IPDI), 4,4′-oxybis (phenylisocyanate), 4,4′-diphenylmethanediisocyanate (MDI), mixtures of MDI and oligomers based on MDI (e.g.Desmodur VL), 2,4-diphenyldiisocyanate, methylene biscyclohexyldiisocyanate (H₁₂MDI), phenylene diisocyanate (p-PDI),trans-cyclohexane-1,4-diisocyanate (CHDI), 1,6-diisocyanatohexane(DICH), 1,5-diisocyanato-naphthalene (NDI), paratetramethylxylenediisocyanate (p-TMXDI) or metatetramethylxylene diisocyanate (m-TMXDI).

An especially preferred isocyanate is MDI or polyisocyanates basedthereon (e.g. Desmodur). Desmodur comprises MDI and oligomers thereofcomprising MDI with CH₂-phenylisocyanate residues. The Desmodur is thusa mixture of various polyisocyanates deriving from MDI. A samplestructure may be

-   -   4,4-methylene bis(phenylisocyanate) 40-50%    -   4,4-methylene bis(phenylisocyanate)+benzylisocyanate: 20-25%    -   4,4-methylene bis(phenylisocyanate)+2 benzylisocyanate: 10%    -   4,4-methylene bis(phenylisocyanate)+3 benzylisocyanate:2%.

(In a reaction like this the product also contains some of the2-isomer). The compound is sold by Shell under the trade name Caradateand under the trade names Hylene and Rubinate by Huntsman.

Preferably two diols should be employed. The diols are preferably usedin a molar ratio of 0.5:1 to 1:0.5, more preferably 0.8:1 to 1:0.8 whentwo diols are used. Preferably no one diol is used in a quantityexceeding 90% mol. of the diol mixture.

Preferred diols include diethylene glycol, tetraethylene glycol andpolyethylene glycols e.g PEG 300, 400 or 600. A preferred diolcombination is diethylene glycol and tetraethylene glycol.

During the coating reaction involving the polyisocyanate, it ispreferred if, in a first stage the polyisocyanate is in excess (e.g.relative to any diol). It is within the scope of the invention to useonly polyisocyante in this step of the coating procedure. This isbelieved to minimise the possibility of gelling occurring during thereaction. Where a large excess of polyisocyante is employed in aninitial coating reaction, it may then be necessary to react, in a secondstage, the coated particles with further diol(s) (e.g. a diol asdescribed above) to react with any unreacted isocyanate groups. Wherethe initial coating reaction uses polyisocyanate alone, it is essentialthat the resulting particle is reacted with at least one diolthereafter.

In such an embodiment, such a diol is preferably a polyethylene glycol.The long chain of the PEG diol allows the formation of a sizable linkerbetween the particle coating surface and hence makes easier reactionwith affinity ligands such as streptavidin.

It is thus within the scope of the invention to react the particles withpolyisocyanate followed by diol, i.e. a stepwise process, to effectcoating.

Typically therefore, the coating reaction may be effected byimpregnating the porous magnetic polymer particle with the epoxides orthe polyisocyanate and diol(s), e.g. using a solution of these (forexample in an organic solvent such as methanol or diglyme) or by mixinga dispersion of the porous particles in an organic solvent with a liquidepoxide or diol/polyisocyanate mixture. Sonication may be used toimprove impregnation and the reaction may be accelerated by raising thetemperature, e.g. to 50-100° C. Any solvent used may be extracted byapplication of sub-ambient pressure.

Generally, the uses to which magnetic polymer particles are put, e.g.their use as diagnostic tools, require an appropriate degree ofelectrophilicity in order that they may participate adequately incoupling and other reactions in aqueous systems prevalent in biologicalmedia.

Whilst the general polarity of the coatings is desirably electrophilic,certain coatings which contain hydrophobic moieties may be incorporatedso as to tailor the degree of electrophilicity to that which is desired.In this way, the invention permits the provision of useful diagnosticand other tools having a wide range of polarities.

If desired, in the process of the invention, the surfaces of the coatedmagnetic polymer particles may be further functionalised, e.g. bycoupling a drug molecule, a reporter label (e.g. a chromophore,fluorophore, enzyme or radiolabel), or a ligand (e.g. an antibody orantibody fragment, a metal ion complexing agent, a member of a specificbinding partner pair (e.g. biotin or streptavidin), an oligopeptide, anoligonucleotide, or an oligosaccharide).

Such coupling may be direct or indirect (and so may or may not involvethe use of a coupling agent to form a linkage between the particle andthe substance being coupled to it) and may be biodegradable ornon-biodegradable. Biodegradable couplings may be desired if themagnetic polymer particles are to be used for the targeted release of anactive compound. Accordingly after coating has been effected, thependent groups of the coating may be manipulated to provide appropriatefunctionality (for example epoxy, hydroxy, amino etc. functionalities)for the attachment of such substances.

The functionalised coated magnetic particle may be bound to an affinityligand the nature of which will be selected based on its affinity for aparticular analyte whose presence or absence in a sample is to beascertained. The affinity molecule may therefore comprise any moleculecapable of being linked to a magnetic probe which is also capable ofspecific recognition of a particular analyte. Affinity ligands thereforeinclude monoclonal antibodies, polyclonal antibodies, antibodyfragments, nucleic acids, oligonucleotides, proteins, oligopeptides,polysaccharides, sugars, peptides, peptide encoding nucleic acidmolecules, antigens, drugs and other ligands. Examples of suitableaffinity ligands are available in the published literature and are wellknown. The use of further binding partners, secondary affinity ligandsand linking groups which is routine in the art will not be discussedfurther herein although it will be appreciated that the use of suchspecies with the particles of the invention is possible if desired.

In an especially preferred embodiment, the particle coating is preparedusing an epoxide compound having a functional group copolymerizable withan acrylate, e.g. a carbon-carbon double bond, for example using two orthree epoxides one of which contains an unsaturated carbon-carbon bond.The coated particles may then be functionalized by reaction with a vinylor acrylic monomer carrying a functional group, for example a carboxylicacid group (e.g. using acrylic acid). Further functionalization may thenreadily be achieved by reaction of the pendant carboxyl groups, e.g. byreaction with N-hydroxysuccinimide or with streptavidin. This processand particles formed by it are novel and form a further aspect of theinvention.

Thus viewed from a further aspect the invention provides coatedpolymeric particles, optionally carrying superparamagnetic crystals,having a coating formed from at least two epoxides, at least one ofwhich having an unsaturated carbon-carbon bond copolymerizable with anacrylic monomer.

Viewed from another aspect the invention provides a process for thepreparation of coated polymer particles, optionally containingsuperparamagnetic crystals, said process comprising reacting porous,surface-functionalized, optionally superparamagnetic crystal-containingpolymer particles with at least two epoxide compounds, at least one ofwhich having an unsaturated carbon-carbon bond copolymerizable with anacrylic monomer; and

-   -   reacting the formed particles with a vinylic monomer, (e.g.        allylglycidyl ether or an acrylic monomer, e.g. acrylic acid or        acrylamide). The resulting particles may then be further reacted        with an affinity ligand e.g. streptavidin. The particles formed        in this process form a still further aspect of the invention.

Viewed from a further aspect the invention provides the use of particlesof the invention in syntheses, extractions or assays, in particular innucleic acid detection.

Epoxides of use in this aspect include glycidylmethacrylate andallylglycidylether.

Introduction of vinyl groups polymerisable with, for example, an acrylicacid can also be achieved by reacting the coating surface with acompound such as methacrylic anhydride. For example, a coated particlecomprising a coating formed from the reaction of two epoxides which iswashed (e.g. in NaOH) to expose hydroxyl functionalities would reactreadily with methyl acrylic anhydride to allow the introduction of vinylgroups to the polymer surface.

As mentioned above, the nature of the external substance coupled to theparticles may be selected on the basis of its ability to bind to aparticular target material. Nucleic acid detection generally involvesprobing a sample thought to contain target nucleic acids using a nucleicacid probe that contains a nucleic acid sequence that specificallyrecognises, e.g. hybridises with, the sequence of the target nucleicacids, such that the nucleic acid affinity ligand and the target nucleicacids in combination create a hybridisation layer. Suitablyfunctionalised particles of the invention, e.g. those coated with atleast two epoxides and carrying a carboxyl group subsequently reactedwith streptavidin, are ideally suited for nucleic acid detection.

Biotinylated single strand oligonucleotide probes bound to streptavidinbeads can be used to isolate sequence specific DNA. The biotinylatedprobes are bound to the beads by mixing the appropriate amount of beadswith an excess of biotinylated probe. The beads/probe are then incubatedwith the DNA sample in a hybridisation buffer, e.g. SSPE or SSC, underconditions appropriate for the length and sequence of the probe and DNA.The excess and unwanted DNA is washed away utilizing the magneticproperties of the beads. The captured DNA can be detected/quantified byPCR etc.

Biotinylated double strand DNA fragments bound to streptavidin beads canbe used to isolate DNA sequence specific binding proteins. Thebiotinylated DNA is bound to the beads by mixing the appropriate amountof beads with an excess of biotinylated DNA fragments. The beads/DNA arethen incubated with the protein sample in a hybridisation buffer, underconditions appropriate for the protein under investigation. The excessand unwanted protein is washed away utilizing the magnetic properties ofthe beads. The captured protein can be eluted from the probe (by highsalt, low salt, heat, low pH etc) for downstream applications anddetection.

The target material may optionally be a material of biological orsynthetic origin, e.g. it may be a molecule or a group of molecules,including for example antibodies, amino acids, proteins, peptides,polypeptides, enzymes, enzyme substrates, hormones, lymphokines,metabolites, antigens, haptens, lectins, avidin, streptavidin, toxins,poisons, environmental pollutants, carbohydrates, oligosaccharides,polysaccharides, glycoproteins, glycolipids, nucleotides,oligonucleotides, nucleic acids and derivatised nucleic acids, DNA, RNA,natural or synthetic drugs, receptors, virus particles, bacterialparticles virus components, cells, cellular components, natural orsynthetic lipid vesicles, polymer membranes, polymer services andparticles and glass and plastic surfaces.

Where the beads of the invention are to be employed in immunoassays ithas surprisingly been found that tosylation of the particles aftercoating results in particles which exhibit improved performance inimmunoassays. Thus, in a preferred embodiment, particles carrying acoating can be tosylated, e.g. by reaction of the particles withtosylchloride in the presence of a base. The resulting tosylated coatedparticles are new and form a further aspect of the invention. By tosylis meant a toluene-4-sulphonyl group.

Moreover, such tosylated species can be readily reacted with affinityligands, e.g. streptavidin to form still further new particles.

Thus viewed from a further aspect, the invention provides coatedpolymeric particles, carrying superparamagnetic crystals, having acoating formed from at least one polyisocyanate and at least one diol,which is subsequently tosylated, e.g. by reaction with tosyl chlorideand optionally then reacted with an affinity ligand, e.g. streptavidin.

Viewed from another aspect, the invention provides coated polymericparticles, optionally carrying superparamagnetic crystals, having acoating formed from at least one epoxide, which is subsequentlytosylated, e.g. by reaction with tosyl chloride, and optionally thenreacted with an affinity ligand, e.g. streptavidin.

Moreover, it has surprisingly been found that particles of the diametersclaimed herein have a greatly increased capacity for binding compared tobeads of greater size, e.g. 3 μm beads. It is envisaged that the bindingcapacity of the claimed beads is over 200% greater than that of largerbeads allowing the use of considerably lower amounts particles in anassay procedure.

The beads of the invention are therefore of utility inadsorption/desorption processes analogously to the mechanisms inReversed Phase chromatography or hydrophobic interaction chromatography.Reversed phase chromatography is a separation technique that utilises ahydrophobic adsorption interaction between a solute molecule (e.g. aprotein) and an immobilised hydrophobic ligand (e.g. the surface ofbeads). This interaction is usually so strong that it can occur insolutions of low ionic strength and is broken by the use of organicsolvents (e.g. acetonitrile). Reversed phase chromatography can be usedto fractionate complex protein samples and for desalting proteinsamples. RPC is usually performed using a solid phase packed in to acolumn. The beads of the invention enable the technique to be performedwithout a column, without sample dilution and to be automated with highthroughput.

Hydrophobic interaction chromatography (HIC) is a separation techniquethat utilises a hydrophobic adsorption interaction between a solutemolecule (e.g. a protein) and an immobilised hydrophobic ligand (e.g.the surface of beads). This interaction is weaker than the interactionsutilised during RPC and requires promotion by high salt concentrations.Consequently, decreasing salt concentrations can be used to break theseadsorption interactions. HIC can be used to fractionate complex proteinsamples and for desalting protein samples. HIC is usually performedusing a solid phase packed in to a column. The beads of the inventionenable the technique to be performed without a column, without sampledilution and to be automated with high throughput.

The invention will now be described further by reference to thefollowing examples. These are not intended to be limitative but merelyexemplary of the invention.

EXAMPLE 1 Preparation of 0.3 μm Seed Particles

1600 g styrene was extracted with 2 L 10 wt. % sodium hydroxide, washedwith water to pH 7 and then flushed with argon for 10 min. In a 10Lreactor, 8000 g of water and 3.07 g of borax were heated to 80° C., and100 g of water was evaporated off to remove oxygen. Then 19.97 g sodiumdecyl sulphate in 200 ml boiled water was charged and stirred for 10min. Then the washed and substantially oxygen-free styrene was chargedand stirred for 15 min. Then 4.80 g potassium peroxodisulphate wascharged in 200 g boiled water. The mixture was kept at 80° C. in anargon atmosphere for 13 hours. A monodisperse suspension of polymerparticles was formed having a particle diameter of 0.3 μm.

EXAMPLE 2 Preparation of 1.0 μm Polystyrene Particles

8860 g of water, 866 g DOP (dioctanoyl peroxide), 433 g acetone and51.96 g of SDS were homogenized for 25 minutes in a two stage MantonGaulin homogenizer at 400 kg/cm5 in the first stage and 100 kg/cm5 inthe second stage. After homogenization, 5164 g of the emulsion werecharged with a seed suspension of monodisperse polystyrene particleshaving a diameter of 0.3 μm from Example 1. 1891 g of seed suspensioncontaining 297.8 g of polymeric particles and 1593 g of water was used.

After stirring for 24 hours at 25° C., 6414 g of the activated seedparticle suspension were charged with an emulsion containing 103400 g ofwater, 131 g of SDS (sodium dodecyl sulphate), 1556 g ofpolyvinylpyrrolidone K-30, 4157 g of 63.2% divinylbenzene, 1060 gstyrene and 11606 g of toluene (as a porogen). The emulsion washomogenized for 25 mins at 400 kg/cm5 in the first stage and 100 kg/cm5in the second stage.

After swelling for 2 hours at 25° C., 46676 g of water were charged tothe reactor and then the dispersion was polymerized for 1 hour at 60° C.and 20 hours at 70° C. A monodisperse suspension was formed having aparticle diameter of 1.0 μm.

The particles were washed with methanol and butylacetate and dried. ByBET the specific surface area was determined to be 570 m5/g drysubstance.

EXAMPLE 3 Nitration

A mixture of 9920 g 95% sulphuric acid and 3020 g 65% nitric acid wascooled to 10° C. and then 400 g 1.0 μm dry porous crosslinkedpolystyrene particles from Example 2 were charged. The temperature wasraised to 30° C. for 1 hour 30 min. The suspension was charged with 60Lice and water. The particles were washed with water and methanol byfiltration. The resulting particles contained 9.0 wt. % nitrogen.

EXAMPLE 4 Incorporation of Iron

2579 g FeSO₄.7H₂O and 3.2 g MnSO₄.H₂O were added to a suspension of 4144g nitrated porous particles from Example 3 containing 450 g of particlesand 3694 g of water. The suspension was stirred at room temperature for30 min. 3285 g of 25% NH3 in water was added. The temperature was raisedto 60° C. for 2 hours. The suspension was cooled and the particles werewashed with water by centrifugation. After purification the particleswere transferred to methanol. Analysis of the particles showed a contentof 330 mg Fe/g DS (dry substance) and 0.9 mg Mn/g DS.

EXAMPLE 5 Coating

133 g of a methanol suspension containing 13.3 g of 1,0 μmstyrene-divinylbenzene polymer particles surface functionalized bynitration and reduction and containing superparamagnetic iron oxide (33wt % Fe) were washed five times with 93 g diglyme. Dry substance ofparticles in diglyme was adjusted to 15 wt % and glycidol (3.07 g),1,4-butanedioldiglycidylether (Araldite DY-026, 8.06 g) andglycidylmethacrylate (5.68 g) was added to the particles. The mixturewas heated to 75° C. and stirred for 20 hours. The particles were thenwashed six times with 70 g methanol and four times with 80 g ofisopropanol.

EXAMPLE 6 Functionalisation with Carboxylic Acid Groups

To 1806 g of a suspension of the particles prepared in Example 5 (425 g)in isopropanol was added methanol (774 g), acrylic acid (514 g) and2,21-azoisobutyronitrile (23.4 g). The mixture was heated to 73-75° C.and stirred for 20 hours. The particles were then washed six times with2338 g methanol and once with 3018 g of 0.15 M NaOH. The particlecontent (dry substance basis) was adjusted to 12 wt. % and the mixturewas heated to 75° C. and stirred for 3.5 hours. The particles werewashed three times with 3188 g water and five times with 3188 g 0.01 MNaOH.

47 g of a suspension of the particles prepared in Step (A) (13.3 g) inisopropanol was added methanol (24 g), acrylic acid (9.64 g) and2,2′-azoisobutyronitrile (0,41 g). The mixture was heated to 73-75° C.and stirred for 20 hours. The particles were then washed six times with73 g methanol and once with 94 g of 0.15 M NaOH. Dry substance of themixture of particles and 0.15 M NaOH was adjusted to 12 wt % and themixture was heated to 75° C. and stirred for 3.5 hours. The particleswas washed with three times with 99 g water and five times with 99 g0.01 M NaOH.

EXAMPLE 7 Functionalisation of Carboxylic Acid Groups toN-Hydroxysuccinimide Ester

50 g of a suspension of 5.0 g of the particles of Example 6 wereacidified by washing with 0.1 M acetic acid (3×50 mL). The acidifiedparticles (which had a carboxylic acid content of 0.5 mmole/g DS) werethen washed with acetone (4×50 mL) and concentrated on a magnet. Extraacetone was added until a total of 35.6 g suspension was achieved.N-hydroxysuccinimide (2.90 g, 25 mmole) and diisopropylcarbodiimide(3.16 g, 25 mmole) were then added. The reaction mixture was stirred atroom temperature for 5 hours. The particles were then washed withacetone (5×50 mL).

EXAMPLE 8 Immobilisation of Streptavidin

1. 20 mg of carboxylic acid functionalised beads of Example 6 weredispersed in 1 ml 0.01 M NaOH.

2. The beads were separated on a magnet and the supernatant discharged.

3. The beads were washed three times with 1 ml 30 mM2-morpholino-ethanesulfonic acid (MES) pH 6.1.

4. 1.8 mg streptavidin dissolved in 900 μl 30 mM MES pH 6.1 was added tothe beads.

5. The beads and streptavidin were incubated on mixing device at roomtemperature for 15 minutes.

6. 3 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) dissolved in75 μl cold distilled water was added.

7. The mixture was incubated on a mixing device at room temperature forone hour.

8. The beads were washed three times with Phosphate buffered saline(PBS) pH 7.4 with 0.01% (w/v) Tween 20 in order to remove excessstreptavidin and EDC.

9. The beads were resuspended in PBS pH 7.4 with 0.1% Tween 20.

The amount of streptavidin was measured by use of tracer amounts ofI¹²⁵-labelled streptavidin during coating. The relative amount ofI¹²⁵-labelled streptavidin gives the amount of streptavidin coated tothe surface.

Free biotin binding capacity was determined by addition of excessC¹⁴-labelled biotin to the streptavidin coated beads. Excess biotin waswashed away and the amount of C¹⁴-biotin was measured using abeta-scintillation counter.

The amount of streptavidin coating was found to be 68 μg per mg beads,and the free biotin binding capacity was 3100 mmol free biotin per mgbeads.

EXAMPLE 9 Step A

To 40.0 g of 1.0 Φm styrenedivinylbenzene polymer particles containingsuperparamagnetic iron oxide (33 wt % Fe) (made analogously to Example4) in 200 g diglyme, 100 g of 1,4-butandiol diglycidyl ether and 100 gof glycidol were added. The reaction mixture was stirred at 250 rpm for20 hours at 90 EC. The particles were then washed five times with 400 mLmethanol and four times with 400 mL deionised water.

Step B

To 72 g of an aqueous suspension of the particles prepared as in Step A(particle content 10 wt %), 119 g of sodium hydroxide was slowly added.169.2 g of allyl glycidol ether was then added. After stirring at 250rpm for 18 hours at 60 EC, the particles were washed five times with1400 mL of methanol.

EXAMPLE 10

To 10 g of 1.0 (m styrenedivinylbenzene polymer particles containingsuperparamagnetic iron oxide (33 wt % Fe)(made analogously to Example 4)in 40 g diglyme, 16.7 g of allyl glycidyl ether, 16.8 g of 1,4-butandioldiglycidyl ether, and 16.8 g of glycidol were added. The reactionmixture was stirred at 300 rpm at 90 EC for 20 hours. The particles werethen washed five times with 100 mL methanol.

EXAMPLE 11

78 g of a diglyme suspension of 13 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) was added DesmodurVL (35.83 g), diethyleneglycol (2.82 g), tetraethyleneglycol (4.84 g).The mixture was heated at 80° C. and stirred for 21 hours.

A mixture of diethyleneglycol (47.04 g), tetraethylene glycol (80.45g)and 1,4-diazabicyclo(2.2.2)octane (1.29 g) was added to the particlesuspension. The mixture was heated at 80° C. and stirred for 2 hours.The particles were cooled and washed three times with 100 g diglyme andfour times with 100 g of acetone.

EXAMPLE 12

22 g of a methanol suspension of 1.0 μm styrene-divinylbenzene polymerparticles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) (made analogouslyto Example 4) were washed five times with 14 g diglyme. Dry substance ofparticles in diglyme was adjusted to 16 wt % and Araldite DY-026(1,4-butanedioldiglycidylether) (26 g) and glycidol (26 g) was added tothe particles. The mixture was heated to 90° C. and stirred for 20hours. The particles were then washed 5 times with 80 g methanol andfour times with 45 g of diglyme.

To 18 g of the diglyme suspension of particles was added methacrylicanhydride (15 g) and pyridine (0.4 g). The mixture was heated to 75° C.and stirred for 20 hours. The particles were then washed five times with90 9 methanol and three times with 60 g of isopropanol.

EXAMPLE 13

124.45 gram of a diglyme suspension of 20 g of 1.0 μmstyrene-divinylbenzene polymer particles surface functionalized bynitration and reduction and containing superparamagnetic iron oxide (33wt % Fe) (made analogously to Example 4) was added2,2-Bis[4-(glycidyloxy)phenyl]propane (Bisphenol A diglyidylether,Araldit LY-564) (42 g). The mixture was heated to 60° C. and stirred for2 hours and the cooled to room temperature and stirred over night. Thesuspension was washed three times with diglyme and dry substanceadjusted to 12%.

Glycidol (4.58 g), Araldite DY-026 (1.4-butanedioldiglycidylether) (12.1g) and glycidylmethacrylate (8.57 g) were added. The mixture was heatedat 75° C. and stirred for 21 hours. The particle susupension was cooledand the particles were washed six times with 100 g methanol and fourtimes with 100 g of isopropanol.

EXAMPLE 14

516 gram of a water suspension of 51.6 g of 1.0 μmstyrene-divinylbenzene polymer particles surface functionalized bynitration and reduction and containing superparamagnetic iron oxide (33wt % Fe) (375 g) was added solid sodium hydroxide (144.89 g), themixture was stirred and temperature kept below 42° C. during theaddition. Allylglycidylether (206.4 g) was added, and the mixture washeated to 60° C. stirred for 18 hours. The particle suspension wascooled and the particles were washed four times with 1500 g methanol.

EXAMPLE 15

90 g of a methanol suspension of 15 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) (made analogouslyto Example 4) was washed three times with 60 g diglyme and2,2-Bis[4-(glycidyloxy)phenyl]propane (Bisphenol A diglyidylether,Araldit LY-564) (70.40 g) and 1,4-butanedioldiglycidylether (AralditDY-026) (4,61 g) added. The mixture was heated at 95° C. for 20 hours.The particles were cooled and washed three times with 100 g diglyme andthree times with 100 g of methanol.

EXAMPLE 16

To 23.48 g of a diglyme suspension of 5 g of 1.0 μmstyrene-divinylbenzene polymer particles surface functionalized bynitration and reduction and containing superparamagnetic iron oxide (33wt % Fe) (made analogously to Example 4) was added2,2-Bis[4-(glycidyloxy)phenyl]propane (Bisphenol A diglyidylether,Araldit LY-564) (23.47 g) and polyethyleneglycoldiglycidylether Mw-300(3.07 g). The mixture was heated at 95° C. for 20 hours. The particleswere cooled and washed four times with 40 g of methanol.

EXAMPLE 17

To 24.46 g of a diglyme suspension of 5 g of 1.0 μmstyrene-divinylbenzene polymer particles surface functionalized bynitration and reduction and containing superparamagnetic iron oxide (33wt % Fe) (made analogously to Example 4) was added2,2-Bis[4-(glycidyloxy)phenyl]propane (Bisphenol A diglyidylether,Araldit LY-564) (23.54 g) and polyethyleneglycoldiglycidylether Mw-500(15.28 g). The mixture was heated at 95° C. for 20 hours. The particleswere cooled and washed five times with 40 g of methanol.

EXAMPLE 18

To 26.82 g of a diglyme suspension of 5 g of 1.0 μmstyrene-divinylbenzene polymer particles surface functionalized bynitration and reduction and containing superparamagnetic iron oxide (33wt % Fe) (made analogously to Example 4) was added2,2-Bis[4-(glycidyloxy)phenyl]propane (Bisphenol A diglyidylether,Araldit LY-564) (46.93 g) and glycerolpropoxylate triglycidylether(69.98 g). The mixture was heated at 95° C. for 20 hours. The particleswere cooled and washed five times with 40 g of methanol.

EXAMPLE 19 Immobilization of Streptavidin

20 mg of coated beads from Example 17 were dispersed in 1 ml 0.1 MNa-phosphate buffer pH 7.4. Beads were separated on a magnet and thesupernatant discharged. This was repeated twice. 1 mg streptavidindissolved in 900 ul 0.1 M Na-phosphate buffer pH 7.4 was added to thebeads. Beads and streptavidin were incubated on a mixing device for 20hours at room temperature. The beads were washed three times with PBS pH7.4 with 0.1% BSA.

The free biotin binding capacity was measured as described in example 20and found to be 1900 pmol free biotin per mg beads.

EXAMPLE 20 Binding Capacity for Biotinylated DNA Molecules

1. Plasmid DNA fragments of 1090, 526, 217 base pairs were biotinylatedand europium labelled by PCR.

2. The excess biotin and europium labels were removed by commercial PCRclean-up techniques and the labelled DNA quantified by optical densityat 260 nm and time resolved fluorescence.

3. Streptavidin coated beads (Example 8) were washed once in a 2 timesconcentrated binding buffer (2M NaCl, 20 mM Tris HCl, 0.2M EDTA pH7.5).

4. 5 μg of the washed beads were resuspended in 100 μl of binding bufferin each well of a 96 well plate.

5. An excess of DNA (0.55 pmol of 1090 bp, 1.1 pmol of 526 bp and 2.2pmol of 217 bp) diluted in 100 μl of water was added to the beads andincubated with gentle shaking for 15-30 minutes at room temperature.

6. The plate is placed on the magnet and the supenatant removed.

7. The beads -DNA complex are washed 3 times with 200 μl of wash buffer(10 mM Tris-HCl pH7.8, 0.01% Tween 20).

8. The beads-DNA complex are resuspended in 200 μl DELFIA Enhancementsolution and incubated, protected from light, with shaking at roomtemperature for 10 minutes.

9. The plate is placed on the magnet and the Enhancement solution istransferred to a FluorNunc 96 well plate and the europium signal ismeasured by time resolved fluorescent (Wallac Victor plate reader) andgiven as counts per second (cps).

10. The DNA bound to the beads is calculated from the percent of cpsadded in 0.55, 1.1 and 2.2 pmol of DNA, that has bound to the beads.

The binding capacity of the beads is approximately 200 pmol/mg for the217 bp fragment, 80-100 pmol/mg for the 526 bp fragment and 35-45pmol/mg for the 1090 bp fragment.

EXAMPLE 21

To 23.8 g of a diglyme suspension of 5 g of 1.0 μmstyrene-divinylbenzene polymer particles surface functionalized bynitration and reduction and containing superparamagnetic iron oxide (33wt % Fe) (made analogously to Example 4) was added Desmodur VL (18.34g), diethyleneglycol (1.45 g), tetraethyleneglycol (2.48 g) and diglyme(36.2 g). The mixture was heated at 80° C. and stirred for 20 hours.

A mixture of polyethyleneglycol Mw˜300 (68.24 g), tetraethylene glycol(41.30 g) and 1,4-diazabicyclo(2.2.2)octane (0.68 g) was added to theparticle suspension. The mixture was heated at 80° C. and stirred for 1hour. The particles were cooled and washed three times with 100 gdiglyme and four times with 100 g of acetone.

EXAMPLE 22 Immobilization of Immunoglobulin

20 mg of coated beads from Example 21 were dispersed in 1 ml 0.1 MBorate buffer pH 9.0. Beads were separated on a magnet and thesupernatant discharged. This was repeated twice. 1 mg mouse IgG1anti-human alpha feto protein dissolved in 900 ul 0.1 M Borate buffer pH9.0 was added to the beads. Beads and antibody were incubated on amixing device for 20 hours at room temperature. The beads were washedthree times with PBS pH 7.4 with 0.1% BSA.

The amount of antibody bound to the beads was measured by the use oftracer amounts of I125-labelled antibody during coating. The relativeamount of labelled antibody bound to the beads gives the total amount ofantibody immobilized. Amount of protein immobilized to the beads was 22μg antibody per mg beads.

The binding of alpha feto protein to the immobilized antibody wasmeasured by addition of diltuions of umbilical cord blood and detectedby use of a second mouse IgG anti-human alpha feto protein labelled withEu³⁺. The labelled antibody was detected with Time Resolved FluorescenceSpectroscopy. A standard curve dilution of the umbilical cord bloodshowed increased signal with increasing concentration.

EXAMPLE 23

26.82 g of a diglyme suspension of 5 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) (made analogouslyto Example 4) was added Desmodur VL (18.34 g), polyethyleneglycol Mw˜600(15.84 g) and diglyme (33.20 g). The mixture was heated at 80° C. andstirred for 20 hours.

A mixture of polyethyleneglycol Mw˜600 (263.82 g) and1,4-diazabicyclo(2.2.2)octane (0.66 g) was added to the particlesuspension. The mixture was heated at 80° C. and stirred for 1 hour. Theparticles were cooled and washed three times with 100 g diglyme and fourtimes with 100 g of acetone.

EXAMPLE 24 Functionalisation with Carboxylic Acid Groups

To 15.87 g of a suspension of the particles prepared in Example 5 (2.5g) in isopropanol was added acrylic acid (1.84 g), acrylamide (1.79 g),methanol (5.95 g) and 2,2′-azoisobutyronitrile (0.28 g). The mixture washeated to 75° C. and stirred for 20 hours. The particles were thenwashed four times with 20 g isopropanol and 4 times with 25 g of 0.15 MNaOH.

EXAMPLE 25 Functionalisation with Carboxylic Acid Groups

To 8.44 g of a suspension of the particles prepared Example 5 (1 g) inwater was added acrylic acid (0.37 g), allylglycidylether (1.34 g),dimethylsulfoxide (0.91 g) and 2,2′-azoisobutyronitrile (0.06 g). Themixture was heated to 75° C. and stirred for 20 hours. The particleswere then washed four times with 10 g isopropanol and 4 times with 10 gof 0.15 M NaOH.

EXAMPLE 26

3.9 g of a diglyme suspension of 2.7 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) (made analogouslyto Example 4) was added hexamethylenediisocyanate (2.5 g),diethyleneglycol (0.5 g) and tetraethyleneglycol (1.0 g). The mixturewas heated at 80° C. and stirred for 20 hours.

A mixture of diethylene glycol (2.65 g), tetraethylene glycol (4.66 g)and 1,4-diazabicyclo(2.2.2)octane (0.07 g) was added to the particlesuspension. The mixture was heated at 95° C. and stirred for 2-3 hours.The particles were cooled and washed four times with 20 g acetone.

EXAMPLE 27

23.8 g of a diglyme suspension of 5 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) (made analogouslyto Example 4) was added Desmodur VL (18.34 g), dietyleneglycol (1.45 g),tetraetyleneglycol (2.48 g) and diglyme (36.2 g). The mixture was heatedat 80° C. and stirred for 20 hours.

A mixture of polyethyleneglycol Mw˜400 (90.03 g), tetraethylene glycol(41.20 g) and 1,4-diazabicyclo(2.2.2)octane (0.68 g) was added to theparticle suspension. The mixture was heated at 80° C. and stirred for 1hour. The particles were cooled and washed three times with 100 gdiglyme and four times with 100 g of acetone.

EXAMPLE 28

23.8 g of a diglyme suspension of 5 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) (made analogouslyto Example 4) was added Desmodur VL (18.34 g), dietyleneglycol (1.45 g);tetraetyleneglycol (2.48 g) and diglyme (36.2 g). The mixture was heatedat 80° C. and stirred for 20 hours.

A mixture of polyethyleneglycol Mw˜600 (135 g), tetraethylene glycol(41.30 g) and 1,4-diazabicyclo(2.2.2)octane (0.66 g) was added to theparticle suspension. The mixture was heated at 80° C. and stirred for 1hour. The particles were cooled and washed three times with 100 gdiglyme and four times with 100 g of acetone.

EXAMPLE 29

23.8 g of a diglyme suspension of 5 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) (made analogouslyto Example 4) was added Desmodur VL (18.34 g), dietyleneglycol (1.45 g),tetraetyleneglycol (2.48 g) and diglyme (36.2 g). The mixture was heatedat 80° C. and stirred for 20 hours.

A mixture of ethyleneglycol (27.29 g) and 1,4-diazabicyclo(2.2.2)octane(0.66 g) was added to the particle suspension. The mixture was heated at80° C. and stirred for 1 hour. The particles were cooled and washedthree times with 100 g diglyme and four times with 100 g of acetone.

EXAMPLE 30

26.82 g of a diglyme suspension of 5 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) (made analogouslyto Example 4) was added Desmodur VL (18.34 g) and etyleneglycol (1.64g). The mixture was heated at 80° C. and stirred for 20 hours. A mixtureof ethyleneglycol (27.29 g) and 1,4-diazabicyclo(2.2.2)octane (0.66 g)was added to the particle suspension. The mixture was heated at 80° C.and stirred for 1 hour. The particles were cooled and washed three timeswith 100 g diglyme and four times with 100 g of acetone.

EXAMPLE 31 Activation with Tosyl Groups

To 17.1 g of an acetone suspension of 4 g of Example 30 were addedtosylchloride (16 g) and pyridine (7.85 g). The mixture was stirred at25° C. for 17 hours. The particles was then washed three times with 50ml acetone, once with 50 ml of a mixture of 80% by weight of acetone inwater, once with 50 ml of a mixture of 60% by weight of acetone inwater, once with 50 ml of a mixture of 30% by weight of acetone in waterand three times with 50 ml water.

EXAMPLE 32 Immunoassay for Alpha Feto Protein

50 μg of immunoglobulin coated beads from example 22 was added to 100 μlof dilutions of umbilical cord blood and the content of Alpha FetoProtein allowed to bind during incubation for 15 minutes. Excessumbilical cord blood was washed away. To this complex a second detectionmouse IgG anti-human alpha feto protein labelled with Eu³⁺ was added.After incubation and removal of excess detection antibody the amount oflabelled antibody was detected with Time Resolved FluorescenceSpectroscopy. A standard curve dilution of umbilical cord blood withknown amont of Alpha Feto Protein showed increased signal withincreasing concentration. By use of this standard curve the amount ofAlpha Feto Protein in samples could be determined.

EXAMPLE 33 Immunoassay for Myoglobulin

Beads coated similarly as in example 22, but with mouse IgG anti-humanmyoglobulin were used to analyse the content of Myoglobulin in humancitrated plasma samples on a Liaison immunoassay instrument. Beads andsamples (10 ul) were incubated for 10 minutes and excess sample washedaway. Detection antibody conjugated with Acridinium orange ester wasadded. After incubation for 10 minutes excess detection antibody waswashed away, and the chemiluminscent signal was developed and detectedin the luminescence reader in the Liaison instrument.

EXAMPLE 34 Immunoassay for D-Dimer

Beads coated similarly as in example 22, but with mouse IgG anti-humanD-dimer were used to analyse the content of Myoglobulin in humancitrated plasma samples on a Liaison immunoassay instrument. Beads andsamples (10 ul) were incubated for 10 minutes and excess sample washedaway. Detection antibody conjugated with Acridinium orange ester wasadded. After incubation for 10 minutes excess detection antibody waswashed away, and the chemiluminscent signal was developed and detectedin the luminescence reader in the Liaison instrument.

EXAMPLE 35 Immunoassay for Intact PTH

Beads coated with streptavdin as in example 19 were used to analyse thecontent of intact PTH in samples of human EDTA plasma on a Liaisonimmunoassay instrument. 150 ul of EDTA plasma was incubated with abiotinylated polyclonal goat antibody raised against intact PTH, and anacridinium ester conjugated polyclonal goat antibody raised similarly toyield an immuncomplex. The streptavidin coated beads was added to theimmuncomplex and incubated to allow the biotin to bind to theimmobilized streptavidin. The beads were washed and the chemiluminescentsignal developed and measured in the luminescence reader in the Liaisoninstrument.

EXAMPLE 36

22 g of a methanol suspension of 1.0 μm styrene-divinylbenzene polymerparticles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) (made analogouslyto Example 4) were washed five times with 14 g diglyme. Dry substance ofparticles in diglyme was adjusted to 16 wt % and glycidylmethacrylate(50 g) and Iron (III) chloride (0.62 g) was added to the particles. Themixture was heated to 75° C. and stirred for 20 hours. The particleswere then washed six times with 14 g methanol and four times with 13 gof isopropanol.

EXAMPLE 37

26.82 g of a diglyme suspension of 5 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) (made analogouslyto Example 4) was Desmodur VL 18.34 g) and diglyme (33.20 g). Themixture was heated at 80° C. and stirred for 20 hours. A mixture ofpolyethyleneglycol Mw˜600 (236.82 g) and 1,4-diazabicyclo(2.2.2)octane(0.66 g) was added to the particle suspension. The mixture was heated at80° C. and stirred for 1 hour. The particles were cooled and washedthree times with 100 g diglyme and four times with 100 g of acetone.

EXAMPLE 38

10.74 g of a diglyme suspension of 2 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) (made analogouslyto Example 4) was added glycidol (6.84 g). The mixture was heated at 90°C. for 20 hours. The particles were cooled and washed eight times with17 g of methanol and four times with 17 g of water.

EXAMPLE 39

14.52 g of a diglyme suspension of 2.50 g of 1.0 μmstyrene-divinylbenzene polymer particles surface functionalized bynitration and reduction and containing superparamagnetic iron oxide (33wt % Fe) (made analogously to Example 4) was added2,2-Bis[4-(glycidyloxy)phenyl]propane (Bisphenol A diglyidylether,Araldit LY-564) (13.03 g). The mixture was heated at 95° C. for 20hours. The particles were cooled and washed three times with 17 g ofdiglyme and five times with 17 g of methanol.

EXAMPLE 40 Binding Capacity for Biotinylated Protein Molecules

1. 5 μg of the streptavidin beads (Ex 19) were washed once in DELFIAassay buffer and mixed with an excess of biotinylated antibody labelledwith europium³⁺ in a 96 well plate.

2. Incubation with mixing at room temperature was carried out for 20minutes to allow the antibody to bind to the beads.

3. The plate was placed on the magnet and the supenatant removed.

4. The beads -antibody complex were washed 3 times with 2001 of DELFIAwash buffer

5. The beads-antibody complex were resuspended in 200 μl DELFIAEnhancement solution and incubated, protected from light, with shakingat room temperature for 10 minutes.

6. The plate was placed on the magnet and the enhancement solution istransferred to a FluorNunc 96 well plate and the europium signalmeasured by time resolved fluorescent (Wallac Victor plate reader) andgiven as counts per second (cps).

7. The amount of antibody bound to the beads was calculated from thepercent of cps added that has bound to the beads.

The binding capacity of the beads was approximately 8.0 μg biotinylatedantibody per mg beads.

EXAMPLE 41

30.1 g of a diglyme suspension of 5 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) was added DesmodurVL (13.8 g), diethyleneglycol (2.08 g). The mixture was heated at 80° C.and stirred for 20 hours. A mixture of diethyleneglycol (35 g) and1,4-diazabicyclo(2.2.2)octane (0.5 g) was added to the particlesuspension. The mixture was heated at 80° C. and stirred for 1 hour. Theparticles were cooled and washed three times with 80 ml diglyme and fivetimes with 80 ml of acetone.

EXAMPLE 42

30.8 g of a diglyme suspension of 5 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (33 wt % Fe) was added DesmodurVL (13.8 g). The mixture was heated at 80° C. and stirred for 20 hours.A mixture of diethyleneglycol (35 g) and 1,4-diazabicyclo(2.2.2)octane(0.5 g) was added to the particle suspension. The mixture was heated at80° C. and stirred for 1 hour. The particles were cooled and washedthree times with 80 ml diglyme and five times with 80 ml of acetone.

EXAMPLE 43

7.0 g of 1.0 μm styrene-divinylbenzene polymer particles surfacefunctionalized by nitration and reduction and containingsuperparamagnetic iron oxide (33 wt % Fe) was adjusted to 20 weightpercent dry material in diethylene glycol dimethylether. 17.5 g AralditDy-026 and 17.5 g Glycidol was added to the suspension, and the mixturewas heated at 90° C. for 20 hours. The beads were washed five times withmethanol (200 ml) and five times with water (200 ml).

EXAMPLE 44

To a 80 g of a suspension of 8.0 g beads in example 43 was added 22.5 gsodium hydroxide and 32 g of allylglycidylether. The beads were stirredand heated at 60° C. for 18 hours, and worked up by washing with fourtimes with 240 methanol.

EXAMPLE 45 Reversed Phase Chromatography (RPC)

The beads of examples 41, 42, 11, 15, and 30 were used to fractionate aprotein mixture consisting of conalbumin, soya bean trypsin inhibitor,alcohol dehdrogenase, cytochrome C and diamine oxidase, or cell lysatesfrom SW 480 cells. 1 mg of beads were pre-washed and resuspended in 10ul of an RPC adsorption buffer (obtained from Bruker Daltonics oralternatively 50 mM sodium phosphate buffer, 1 M ammonium sulphate, 0.1%trifluoroacetic acid) and incubated with 25 ug of the protein mixture orcell lysate for 1 minute. Following adsorption, the beads were washedthree times with washing buffer (obtained from Bruker Daltonics or 50 mMsodium phosphate buffer, 1 M ammonium sulphate, 0.1% trifluoroaceticacid) and the proteins subsequently desorbed in fractions by the use ofdesorption buffers containing increasing concentrations of acetonitrile(0, 15, 30, 40 & 50%)

EXAMPLE 46 Hydrophobic Interaction Chromatography (HIC)

The beads of Example 44 were used to fractionate a protein mixtureconsisting of conalbumin, soya bean trypsin inhibitor, alcoholdehdrogenase, cytochrome C and diamine oxidase. 1 mg of beads werepre-washed and resuspended in adsorption buffer (50 mM sodium phosphatebuffer pH 5.8, 1 M ammonium sulphate) and incubated with 25 g of theprotein mixture for 1 minute. Following adsorption, the beads werewashed three times with an appropriate washing buffer (50 mM sodiumphosphate buffer, 1 M ammonium sulphate) and the proteins subsequentlydesorbed in fractions using desorption buffers containing decreasingconcentrations of ammonium sulphate (0.8, 0.6, 0.4, 0.2 & 0.0 M).

EXAMPLE 47

26.81 g of a diglyme suspension of 5 g of 1.0 μm styrene-divinylbenzenepolymer particles surface functionalized by nitration and reduction andcontaining superparamagnetic iron oxide (46 wt % Fe) (375 g) was addedDesmodur VL (18.39 g) and diglyme (33.50 g). The mixture was heated at80° C. and stirred for 20 hours. The particles were cooled and washedthree times with 100 g of dimethylformamide and added 2,2(ethylenedioxide)-diethylamin (74.4 g) and 1,4-diazabicyclo(2.2.2)octane(0.63 g). The mixture was heated to 80° C. for two hours. The particleswere cooled and washed four times with 100 g dimethylformamide.

6.66 g of this dimethylformamide suspension was added1,4-butanedioldiglycidylether (Araldit LY-026) (3.03 g). The mixture washeated to 70° C. for 20 hours. The particles were cooled and washedthree times with 17 g of dimethylformamide.

1-12. (canceled)
 13. A process for the preparation of coated polymerparticles containing superparamagnetic crystals, said process comprisingreacting porous, surface-functionalized, superparamagneticcrystal-containing polymer particles of diameter 0.5 to 1.8 μm with atleast one epoxide compound.
 14. A process as claimed in claim 13 whereinone epoxide is used, said epoxide being glycidyl methacrylate, saidreaction being effected in the presence of iron (Ill) chloride.
 15. Aprocess as claimed in claim 13 wherein at least two epoxides are used.16. A process as claimed in claim 15 wherein said at least two epoxidecompounds comprise at least one bisepoxide compound.
 17. A process asclaimed in claim 16 wherein said epoxide compounds comprise glycidol and1,4-bis-(2,3-epoxypropoxy)butane.
 18. A process as claimed in claim 16wherein said epoxide compounds comprise 1,4-bis-(2,3-epoxypropoxy)butaneand 2,2-bis(4-(2,3-epoxypropoxy)phenyl)-propane.
 19. A process asclaimed in claim 15 wherein said epoxide compounds comprise glycidol,allylglycidyl ether and 1,4-bis-(2,3-epoxypropoxy)butane.
 20. A processas claimed in claim 15 wherein one of said epoxide compounds is apolyethylene glycol diglycidylether with a molecular weight of 150 to1000 g/mol.
 21. A process as claimed in claim 13 for the preparation ofcoated polymer particles, containing superparamagnetic crystals, saidprocess comprising reacting porous, surface-functionalized,superparamagnetic crystal-containing polymer particles with at least twoepoxide compounds, at least one of which having an unsaturatedcarbon-carbon bond copolymerizable with an acrylic monomer; and reactingthe formed particles with an acrylic monomer.
 22. A process as claimedin claim 13 wherein said particles are also reacted with a polymercross-linking reagent.
 23. A process as claimed in claim 22 wherein saidcrosslinking agent is divinylbenzene.
 24. (canceled)
 25. A process asclaimed in claim 13 wherein the particles are amine functionalised. 26.(canceled)
 27. A process as claimed in claim 13 wherein said poroussurface-functionalized polymer particles are nitrated and reducedstyrene-divinylbenzene polymer particles.
 28. A process as claimed inclaim 13 wherein the diameter of the polymer particles is between 0.5 to1.2 μm.
 29. A process as claimed in claim 14 wherein the diameter of thepolymer particles is about 1 μm.
 30. (canceled)
 31. A process as claimedin claim 13 in which said coated particle is subsequently tosylated. 32.A process as claimed in claim 13 wherein subsequent to the coatingreaction, said particles are coupled to a drug molecule, reporter moietyor ligand.
 33. A process as claimed in claim 32 wherein said ligand is amonoclonal antibody, polyclonal antibody, antibody fragment, nucleicacid, oligonucleotide, protein, oligopeptide, polysaccharide, sugar,peptide, peptide encoding nucleic acid molecule, antigen or drug.
 34. Aprocess as claimed in claim 32 wherein said ligand is streptavidin. 35.A process as claimed in claim 34 said process comprising reactingporous, surface-functionalized, superparamagnetic crystal-containingpolymer particles with at least two epoxide compounds, at least one ofwhich having an unsaturated carbon-carbon bond copolymerizable with anacrylic monomer; reacting the formed particles with an acrylic monomer;and reacting the formed particle with streptavidin.
 36. Coated polymericparticles, optionally carrying superparamagnetic crystals, having acoating formed from at least two epoxides, at least one of which havingan unsaturated carbon-carbon bond copolymerizable with an acrylicmonomer.
 37. A particle obtainable by the process of claims
 13. 38-40.(canceled)